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Abstract Six new strains of Alcaligenes enriched for and isolated as nickel-resistant bacteria resemble Alcaligenes eutrophus H16 and contain both an NAD-reducing, tetrameric soluble hydrogenase and a membrane-bound hydrogenase. None of the soluble hydrogenases share with the Rhodococcus opacus MR11 enzyme tetramer the property of being cleaved easily into two dimeric moieties [a hydrogenase (βδ) and an NADH:acceptor oxidoreductase (αγ)], in the absence of nickel or at low ionic strength. The soluble hydrogenase of the newly isolated strain MR22 of R. opacus equalled that of strain MR11. The absence of a membrane-bound hydrogenase in Alcaligenes denitrificans strain 4a-2 and in Alcaligenes ruhlandii was confirmed.

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Summary 1. Using cells of Hydrogenomonas H 16 after mutagenesis by nitrous acid about 0.4 percent and by 1-nitroso-3-nitro-1-methylguanidine 1.5 percent auxotrophic mutants were found among the survivors. In order to increase the mutant yield an enrichment method was elaborated. The penicillin technique was modified by replacing penicillin by colistine, since penicillin is ineffective for Hydrogenomonas H 16. The incubation of 108 cells/ml under growth conditions in the presence of 50 μg colistine/ml for 10 hrs was found effective; during a model experiment 88 percent of the non-growing auxotrophic cells and only 0.09 percent of the growing wildtype cells survived. 2. 214 mutants were isolated and nutritionally characterized. In addition to auxotrophs many mutants were isolated which lack the ability to utilize fructose. 3. Some of the strains requiring isoleucine, or isoleucine+valine were characterized. On the basis of results of crossfeeding experiments and of enzyme assays it is concluded that the biosynthetic pathways for isoleucine and valine in Hydrogenomonas are the same as in other bacteria.

Notes:
Summary Threonine desaminase and acetohydroxyacid synthase in cell-free extracts of Hydrogenomonas H 16 and mutants derived therefrom have been studied with respect to their regulatory properties. 1. The activity of threonine desaminase has been determined employing two combined optical tests using the oxidation of NADH2 as indicator system: by trapping the ammonia produced through glutamate dehydrogenase and α-ketoglutarate and by reducing the α-ketobutyrate produced through lactate dehydrogenase. Optimum pH has been found to be 9.0 in tris-buffer. The desaminase reaction is completely inhibited already by 0.3 mM L-isoleucine. The inhibition is partially relieved by L-valine. The substrate (threonine) saturation curve has paraboloid shape; in the presence of isoleucine (0.05 mM) its shape is sigmoid. The Hill-coefficient in the absence of the inhibitor is n=1.1 and in the presence of L-isoleucine is n=2.4 (pH 8.3). 2. A prototrophic revertant has been isolated from an isoleucine requiring mutant defective in threonine desaminase. This revertant excretes isoleucine. The threonine desaminase of this revertant differs from the wildtype enzyme by a diminished affinity for isoleucine; half maximal inhibition occurs at 2.6 mM isoleucine in contrast to 0.2 mM at the wildtype enzyme. 3. The acetohydroxyacid synthase has its pH optimum at 9.0. At this pH the inhibition by L-valine amounts to 25%. At pH 7.4 a 50% inhibition was observed at a 0.1 mM concentration of valine. By varying the magnesium concentration an 80% inhibition was observed at 0.2 mM valine. The sensitivity of the enzyme is specific for L-valine; L-isoleucine and L-leucine neither decrease the activity nor act antagonistically. 4. When an isoleucine-valine-auxotrophic mutant was grown in a chemostat and when growth was limited by the concentration of valine, the formation of both enzymes, threonine desaminase and acetohydroxyacid synthase, became derepressed. When growth was limited by the concentration of isoleucine only threonine desaminase became derepressed.

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Abstract Recently isolated coryneform hydrogen bacteria were investigated under taxonomical aspects. Strains 7 C, RH 10, and 14 g are characterized by the snapping type of cell division, 68.5 to 69.7% GC content, dl-diaminopimelic acid in the cell wall, content of metachromatic granules, weak utilization of sugars and inhibitory effect of citrate. The strains are placed to the group 1—genus Corynebacterium—of the classification of coryneform bacteria of Yamada and Komagata (1972) and the name Corynebacterium autotrophicum sp.nov. is proposed. Strains 11 X and RH 12 are characterized by the bending type of cell division, a GC content of 70.2 and 70.5%, ll-diaminopimelic acid in the cell wall, absence of metachromatic granules, utilization of several sugars and no changes in cell morphology by citrate. The strains have to be placed to group 6 of coryneform bacteria.